The isolated perfused heart is a useful model for studying cardiac mechanics, metabolism, and electrophysiology. Its critical disadvantage is that crystalloid-based perfusate, such as Krebs-Henseleit solution (KHS), does not maintain adequate myocardial oxygenation, especially when metabolic demand is high. Perfluorocarbons (PFC) were a popular artificial O2 carrier and were actively studied for clinical use, although development has mostly ceased due to safety concerns. The loss of commercial sources of PFC emulsions detrimentally impacts the scientific community because PFC are particularly valuable for perfused heart studies and tissue engineering approaches. There is an unmet need to demonstrate that individual laboratories can make a PFC emulsion in-house, with the goal of enabling increasingly sophisticated hypotheses of cardiac disease to be tested in adequately oxygenated perfused heart preparations. Additionally, an oxygen-rich perfusate that is compatible with fluorescence imaging, including optical mapping, will dramatically elevate the physiological significance of such studies, especially as optical mapping systems evolve to enable mapping in contracting hearts -- preparations that have a very high oxygen demand. Demonstrating that PFC perfusate maintains myocardial oxygenation and provides oxygen reserve while not substantially interfering with visible-light optical assessments of myocardial function will surely establish a new paradigm for excised heart studies. Our objective is use a custom-made PFC emulsion to provide oxygen reserve in perfused heart studies and reestablish physiological coronary flow rates, such that myocardial metabolism, in-situ mitochondrial function, and electrophysiology can be studied without physiological artifacts associated with inadequate oxygenation, such as KATP channel activation and vasodilation. We will also use a novel motion-tracking optical mapping approach to elucidate the effects of PFC-based oxygenation on sarcolemmal KATP channel activation (via APD changes), heart rate, and conduction velocity in fully working isolated rabbit heart preparations. Our primary goal is to rigorously show that a PFC-based perfusate and optical mapping with motion tracking is a powerful combination for studying energetics and electrophysiology in excised hearts. The first Aim is to determine the PFC concentration (weight/vol) that is required to maintain myoglobin oxygenation and oxidation of the ETC for excised hearts that are arrested, contracting, and working. The second Aim is to measure the effect of PFC on the fluorescence signal attenuation of several common fluorophores, including di-4-ANEPPS, RH237, Rhod- 2AM, and BCECF. The third Aim is to optically map changes in APD and CV before and after PFC perfusion in excised biventricular working hearts. Approaches developed in this project could be applied in any perfused organ experiment, having specific and substantial impact on basic whole-heart research.